Fitoterapia 82 (2011) 1215–1221

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Fitoterapia
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HPLC quantification of coumarin in bastard balm

(Melittis melissophyllum L., Lamiaceae)
F. Maggi a, L. Barboni b,⁎, G. Caprioli a, F. Papa a, M. Ricciutelli c, G. Sagratini a, S. Vittori a
a
School of Pharmacy, University of Camerino, Via Sant'Agostino 1, I-62032 Camerino, Italy
b
School of Science and Technology, Chemistry Division, University of Camerino, Via Sant'Agostino 1, I-62032 Camerino, Italy
c
HPLC-MS laboratory, University of Camerino, Via Sant'Agostino 1, I-62032 Camerino, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Coumarin occurs in many plants used as flavoring and is known to possess hepatotoxic effects.
Received 20 June 2011 Despite in the EFSA ‘Compendium of botanicals containing toxic substances’ coumarin is
Accepted in revised form 10 August 2011 reported to be present in Melittis melissophyllum (bastard balm), a plant traditionally used as
Available online 6 September 2011 beverage in Italy and Serbia, to the best of our knowledge quantitative data has never been
reported. Thus, the amount of coumarin in bastard balm leaves and its variation during the an-
Keywords: nual phenological cycle were determined. The subsp. melissophyllum resulted to contain high
Bastard balm (Melittis melissophyllum) levels of coumarin (14,392 mg/kg), mainly in the early stages of the plant cycle, suggesting
Coumarin prudence in its use as beverage. Furthermore, coumarin was found to be useful as marker com-
Phenological cycle
pound to differentiate the bastard balm subspecies occurring in Italy, since the subsp. albida
HPLC-DAD-MS
contained a much lower content of this molecule (19–34 mg/kg).
Subsp. melissophyllum
Subsp. albida © 2011 Elsevier B.V. All rights reserved.

1. Introduction Synthetic coumarin was used as food flavoring until 1954,

Coumarin (1,2-benzopyrone), firstly isolated in 1822 from
Tonka beans [Dypterix odorata (Aubl.) Willd., also known as
Coumarouna odorata Aubl.] [1], is the parent compound of
a large class of naturally occurring phenolic constituents having
fused benzene and α-pyrone rings. For its strong fragrant
odor, coumarin is widely used in industry as sweetener and
fixative of perfumes, flavor enhancer, and other applications
[2].
Coumarin occurs in many plants and in the corresponding
essential oils used as flavoring ingredients [3–6]. Concerning
plants for food consumption, significant concentrations, up to
25,000 mg/kg, were found in cassia cinnamon [7].
Coumarin is stored in the vacuoles of fresh plant as o-
coumaric acid glucoside, from which is produced by enzymatic
hydrolysis and successive lactonisation, as a consequence of
plant tissues damage during harvesting and processing [8].
⁎ Corresponding author. Tel.: + 39 0737402240; fax: + 39 0737402297.
E-mail address: luciano.barboni@unicam.it (L. Barboni).
when it was banned because of its hepatotoxicity [9,10]. In
Europe, maximum levels for coumarin in foodstuffs are in
the range of 5–50 mg/kg [11], and the tolerable daily intake
(TDI) for coumarin is 0.1 mg/kg body weight, based on the
no observed adverse effect level (NOAEL) for hepatotoxicity
in animals [12] and human data available from the pharma-
ceutical use of the substance [13].
Coumarin has been found in bastard balm (Melittis
melissophyllum L., Lamiaceae) [14], a perennial herb growing
in thermophilous copse broadleaf woodlands. M. melissophyllum
occurs in Italy with two subspecies: subsp. melissophyllum L.,
growing mainly in the north-western and central Italy, and
subsp. albida (Guss) P.W. Ball, living mainly in southern Italy
and islands [15].
In the folk medicine of central Italy the plant, known also
with the local names of “Erba Lupa”, “Erba Limona” or “Cedrina”,
was used as antispasmodic, against insomnia and eye inflamma-
tions [16]. In the Abruzzo region it is still used during transhu-
mance by shepherds who drink a warm decoction as digestive
and to treat cough and sore throat [17]. In Serbia it was used
for its sedative properties, for nervous anxiety, insomnia, and

0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2011.08.011
1216 F. Maggi et al. / Fitoterapia 82 (2011) 1215–1221
hysteria [18]. Because of these properties, people used to make
an aromatic tea with the fresh or dry leaves, to be drunk after
meals as digestive and antispasmodic.
Although coumarin is reported to be present in bastard
balm in the EFSA ‘Compendium of botanicals that have been
reported to contain toxic, addictive, psychotropic or other
substances of concern’ [19], no information is available re-
garding the levels of this molecule, the plant part of possible
concern, and the changes in content during the phenological
cycle.
Therefore, in the present work an HPLC analysis of the
coumarin content in bastard balm leaves has been carried
out. The endogenous levels of total coumarin in one popula-
tion of bastard balm have been monitored, in order to estab-
lish if there is a link between the chemical variability of
coumarin levels and the development stages during the an-
nual phenological cycle. In addition, the quantitative changes
during the plant processing (e.g. evaporation etc.) have been
determined by analyzing both fresh and dry samples. Because
in some plants coumarin is known to occur mainly as a gluco-
sylated precursor (o-coumaric acid glucoside), an additional
aim of this study was to detect this precursor in the plant ex-
tracts by using HPLC-MS. Finally, coumarin was investigated
as a chemotaxonomically useful marker compound to distin-
guish the two bastard balm subspecies occurring in Italy.
2. Experimental procedures
2.1. Plant material
M. melissophyllum subsp. melissophyllum was collected in a
population growing wild in central Italy (Camerino) at differ-
ent stages of the plant phenology (3 May, 18 May, 30 May, 14
June, 28 June, 12 July and 2 August 2010), from the beginning
of vegetative stage to the end of reproductive stage, in order
to establish if there is a link between the variability of couma-
rin levels and the development stages during the annual bio-
logical cycle. To avoid the daily variations, the sampling was
done as far as possible in the same time of the day (from
11 am to 1 pm). M. melissophyllum subsp. albida was collect-
ed in June–July 2010 in three sites in southern Italy and Sicily
(Potenza, Cosenza and Palermo, respectively). Voucher speci-
mens were identified by Dr. Maggi using available literature
and deposited in the Herbarium Camerinensis (CAME) and in
the Herbarium of Centro Ricerche Floristiche dell'Appennino
(APP) (both acknowledged in the Index Herbariorum, http://
sciweb.nybg.org/science2/IndexHerbariorum.asp) of the School
of Environmental Sciences, University of Camerino (Italy)
Table 1
Collection localities of the investigated bastard balm (Melittis melissophyllum) samples.
Subspecies Collection site
melissophyllum Piedilapiaggia (Camerino)
albida Monte Vulture (Potenza)
albida Celico (Cosenza)
albida Madonie, Monti Balatelli (Palermo)
a
Accession number in: CAME, Herbarium Camerinensis, School of Environmental Sciences, University of Camerino, Camerino, Italy; APP, Herbarium of Centro
Ricerche Floristiche dell'Appennino, Barisciano, Italy.
(collection localities and voucher codes are reported in
Table 1).
2.2. Sample preparation
Fresh leaves were manually excised from plants and then
stored in the dark and at room temperature (~22 °C) until
completely dry. Part of material (only for subsp. melissophyllum)
was deep frozen at −20 °C just after its collection and was kept
at freezing temperature until extraction, in order to find out the
potential changes of coumarin content depending on the water
content. Dry leaves were ground to fine powder in a mill (blend-
er MFC model DCFH 48 IKA-WERK, Staufen, Germany), and
sieved (1 mm pore diameter). Fresh leaves were homogenized
with 10 ml of liquid nitrogen for 5 min and reduced into a pow-
der in a mortar. In order to estimate the water content, fresh
leaves, were left in a stove at 110 °C for 24 h. Moisture content
was determined by the weighing of the leaves of each collection
before and after drying. The experiment was performed three
times. Moisture levels in percentages ranged from 81% at the be-
ginning of vegetative stage (3 May 2010) to 60% at the end of
vegetative stage (2 August 2010).
2.3. Reagents and materials
Coumarin (≥99%), methanol (HPLC grade) and acetoni-
trile (HPLC grade) were purchased from Sigma-Aldrich
(Milan, Italy); hydrochloric acid (37%) was obtained from
Carlo Erba (Milan, Italy). Individual stock solutions were pre-
pared by dissolving 1 g of coumarin in 100 ml (ppm) of
methanol and storing this solution in glass-stopped bottles
at 4 °C. Deionized water (N8 MΩ cm resistivity) was obtained
from a Milli-Q SP Reagent Water System (Millipore, Bedford,
MA). Disposable Minisart SRP4 filters with a pore width of
0.45 μm (Chromafil PET-20/25) were from Sartorius Stedim
Biotech GmbH (Goettingn, Germany).
2.4. Extraction procedure
Powdered fresh (2 g) and dry leaves (0.4 g corresponding
to 2 g of fresh leaves taking into account a mean water content
of 80%) were placed in a round bottom flask, equipped with a
magnetic stirrer, and extracted with 5 ml methanol/water
(80:20 v/v) for 30 min at room temperature. Then extracts
were centrifuged at 6500 rpm for 10 min. The methanol/
water mixture was selected as the most effective for extraction
of coumarin according to previous results [3]. For quantifica-
tion of total coumarin, an acidic hydrolysis of the coumarin pre-
cursor was performed according to reported procedures [4,6].
Date of collection GPS coordinates Altitude (m) Voucher codesa
May-August 2010 N 43°09′10″; E 13°07′18″ 590 CAME 13,430
6 July 2010 N 40°56′08″; E 15°37′04″ 1300 APP 42,031
30 June 2010 N 39°18′33″; E 16°23′02″ 1200 APP 42,035
4 June 2010 N 37°54′29″; E 13°59′45″ 933 APP 42,032
 F. Maggi et al. / Fitoterapia 82 (2011) 1215–1221
Briefly, one-fifth of the methanol/water extracts was stirred
in hydrochloric acid (2.5 N, 5 ml) at 80 °C for 90 min. In all
cases, samples were filtered through disposable Minisart
SRP4 (Sartorius Stedim Biotech GmbH, Goettingn, Germany)
filters in PTFE (0.45 μm), and injected (5 μl) into the HPLC
system.
2.5. HPLC-DAD and HPLC-MS analysis
The HPLC system consisted of an Agilent Technologies
(Palo Alto, CA) HP-1100 series, made of an autosampler and
a binary solvent pump, with a diode array detector (DAD).
LC separation was performed on a reversed phase column
Purosfer© C18 (150× 4.6 mm i.d., 5 μm) from Merck KGaA
(Darmstadt, Germany) thermostatted at 40 °C, using mobile
phase A (water with 1% v/v formic acid) and mobile phase B
(acetonitrile) in a gradient program with a flow of 1.0 ml/
min: 0–20 min: 10% B; 20–45 min: 90% B. The volume of a sin-
gle injection was 5 μl. After each injection, column was equili-
brated for 15 min. For quantitative analysis, the wavelength
with the highest intensity was used (278 nm). Furthermore,
UV/Vis spectra between 235 and 600 nm were recorded to
verify the peak identity of coumarin and the peak purity. The
same chromatographic conditions reported above were used
for HPLC-MS (single quadrupole) analysis, performed by a
Hewlett Packard (Palo Alto, CA, USA) HP-1090 Series II, made
of an autosampler and a binary solvent pump, with a mass
spectrometer detector (MSD) equipped with an ESI interface
in negative ionization (NI) mode. The optimized parameters
were: drying temperature, 325 °C; nebulizer gas (nitrogen)
pressure, 60 psi; drying gas (nitrogen) flow rate, 13 l/min;
temperature, 350 °C; and capillary voltage, 3500 V. Quantita-
tive data were collected using Chemstation for LC 3D systems
software rev. B.01.03[204].
2.6. Method validation
An eight point calibration curve was constructed by ana-
lyzing various amounts (2, 10, 50, 100, 500, 1000, 2000 and
3000 mg/l) of coumarin (n = 3 each). It showed a good line-
arity in the whole range of the tested concentrations with a
regression coefficient (R 2) greater than 0.9999. The limits of
detection (LOD) and quantification (LOQ) were 0.17 mg/l
and 0.56 mg/l, respectively.
Among several procedures adopted for the extraction of
coumarin from plant material, we chose that appearing the
best, i.e. using methanol (80%) with magnetic stirring for
30 min at room temperature [3]. This procedure is currently
included in the draft of an official German standard method
for the determination of coumarin in foods. To evaluate
whether the extraction procedure was sufficient for quantify-
ing coumarin in bastard balm leaves, known amounts of the
coumarin standard were added to fresh and dry samples at
two fortification levels (200 and 2000 mg/kg), and the recov-
ery was calculated. It was found to be 104 and 90% for fresh
samples, and 96 and 93% for dry samples at the two fortifica-
tion levels, respectively, with a coefficients of variation (CV%)
below 7% (n = 3) in all cases. The CV values for intra-assay
precisions (n = 4) ranged from 6.7 to 16.2% and from 0.3 to
2.9% for total coumarin in fresh and dry samples, respectively.
1217
These data indicated a good repeatability, since they are
below the recommended limits [20].
2.7. Extraction and MS/MS analysis of the o-coumaric acid
glucoside precursor
There is evidence that o-coumaric acid glucoside is present
as a glycosidically-bound precursor of coumarin in some
plants [8]. To investigate its presence in bastard balm leaves,
the methanol/water extract was purified as follows: part of
the dried extract was suspended in water, washed twice
with n-hexane and ethyl acetate, and extracted twice with
water-saturated n-butanol. The butanolic fraction was then
evaporated to dryness, dissolved in methanol/water 50:50
(100 μg/ml), filtered through 0.45 μm disposable Minisart SRP4
filters, and used for the characterization with FIA-ESI-IT mass
spectrometry by an Agilent (Santa Clara, CA, USA) MSD Trap SL
equipped with an ESI source operating in negative ionization
mode. The operating ESI conditions were: drying gas tempera-
ture, 350 °C; nebulizer gas (nitrogen) pressure, 30 psi; drying
gas (nitrogen) flow rate, 8 l/min; and capillary voltage, 3500 V.
The mass spectrometer was used in the Smart Tune mode of
the using Daltonic Esquire Control software, while infusing the
extracted solution via a syringe pump at a flow rate of 5 μl/min.
The acquisition was in full scan and in Manual MS mode; nega-
tive ions were detected using the normal resolution and the
trap parameters were set in Ion Charge Control (ICC) using
rolling averaging set at 5 with a target of 30,000, and maximum
accumulation time of 300 ms at m/z range from 50 to 1500.
2.8. Statistical analysis
One-way ANOVA, performed by SPSS (v. 13.0) software
package for Windows (SPSS Inc., Chicago, IL, USA), was used to
estimate overall significance. A probability level of 5% (pb 0.05)
was considered significant.
3. Results and discussion
3.1. Identification of coumarin and its glucosylated precursor
Plants have been reported to liberate coumarin from its
precursor by enzyme action after disrupture of cells [21] oc-
curring during harvesting and processing. In the applied pro-
cedure this happened especially after nitrogen treatment and
disruption during plant processing.
Fig. 1 reports the HPLC-DAD chromatograms of fresh
leaves before (a) and after (b) acidic hydrolysis. It can be
seen as coumarin (r. t. 17.0 min, identified by comparison
with authentic coumarin) and its precursor, o-coumaric acid
glucoside (r. t. 7.3 min), were efficiently separated within
17 min. In particular, the peak with retention time of
7.3 min showed a UV spectrum similar to that of coumarin,
although there is a difference in the absorbance range be-
tween 310 and 320 nm (Fig. 2a). Moreover, HPLC-MS analy-
sis showed that the mass spectrum referred to the peak
with retention time of 7.3 min (Fig. 2b), presents two princi-
pal signals at m/z 325.1 and 651.2, that correspond to the [M-
H] − and [2 M-H] − ions. At the beginning of work, these sig-
nals have been hypothesized to be typical of o-coumaric
acid glucoside.
1218 F. Maggi et al. / Fitoterapia 82 (2011) 1215–1221

Fig. 1. HPLC-DAD chromatograms of bastard balm samples (collection of 14

June): a) fresh leaves; b) fresh leaves after acidic hydrolysis.

To confirm the identity of this molecule, the glucosidic frac-

tion was purified according to the procedure reported above,
and analyzed by ESI-MSn obtaining the spectra shown in
Fig. 3. The MS 2 of m/z 325 [M-H] − gave two peaks at 163 and
119 m/z, corresponding to the successive loss of one glucose
and one CO2 units, respectively. The MS 3 of m/z 163 gave a
peak at 119 m/z confirming the above fragmentation pattern.
On this basis and according to the literature [6], the compound
was therefore identified as o-coumaric acid glucoside. In the
present work, the isomeric form in which the precursor occurs
in bastard balm leaves was not investigated.
Fig. 2. a) UV spectra of coumarin and its glucosidic precursor; b) mass spec-
3.2. Total coumarin content in fresh and dry bastard balm leaves trum of glucosidic precursor of coumarin in negative ionization mode using
and changes during the phenological cycle single quadrupole.

M. melissophyllum has been included in the “EFSA Com-

pendium of botanicals that have been reported to contain
toxic substances” [19] for the occurrence of coumarin. How-
ever, no information is available regarding the levels of this
molecule, as well as the plant part with the highest concen-
tration. On the other hand, seasonal changes and environ-
mental conditions may affect the production of secondary
metabolites in various parts of the plant. Therefore, besides
the first quantification of coumarin levels in bastard balm
leaves, in the present study the changes in content during
the phenological cycle of the plant, as well as those occurring
during processing of plant material, have been reported
(Table 2).
The analytical data obtained as reported in the experi-
mental section were corrected for the moisture content and
were expressed on a dry weight basis. Liberation of coumarin
was initiated during sample manipulation, by nitrogen treat-
ment and homogenization of fresh leaves and by shredding of
dry leaves. Consequently, cells were mechanically destroyed,
enabling the enzymes such as glucosidase to interact with the
substrate, and leading to the production of coumarin from
glucosidic precursor. In addition, in order to quantify the
total coumarin occurring in the plant matrix, the glucosidic
precursor was completely hydrolysed, as described above.
The levels of total coumarin in bastard balm samples var-
ied between 4548 mg/kg and 14,392 mg/kg in fresh samples,
and between 3091 mg/kg and 11,125 mg/kg in dry samples
(Table 2). There was a similar pattern in coumarin levels dur-
ing the phenological cycle both in fresh and dry material. The
maximum levels (14,392 mg/kg and 11,125 mg/kg in fresh
and dry leaves, respectively) were observed at the beginning
of cycle (3 May) when the plant shows a high growth rate
(renewal after winter dormant). As a consequence, also the
water content of the plant leaves resulted the maximum
(above 80%), allowing cell vacuoles to contain higher amounts
of the hydrophilic coumarin precursor. This might explain the
role of coumarin as phytoalexin: since the plant at the begin-
ning of the cycle is quite susceptible to wounds or attacks by
other organisms, it produces coumarin as an antipathogenic
 F. Maggi et al. / Fitoterapia 82 (2011) 1215–1221 1219

Fig. 3. a) Fragmentation pattern proposed for the glucosidic precursor of coumarin; b), c) and d) mass spectra (MS, MS2 and MS3) obtained in the ion trap.

compound. Furthermore, the primary site of synthesis of cou- the fruiting (14 June) when it reached the concentration of
marin is suggested to be the young, actively growing leaves. 9742 mg/kg and 6297 mg/kg in fresh and dry leaves, respec-
The above values resulted among the highest detected in tively. After this stage, the coumarin content decreased, and
plant matrices so far, but substantially lower than the published reached the lowest values (4588 mg/kg and 3143 mg/kg in
high contents, ranging between 16,000 and 25,000 mg/kg, fresh and dry leaves, respectively) at the later stages of cycle
detected in cassia cinnamon [7]. The total coumarin content de- (2 August) when the plant lost significant amount of water
creased at early stages of flowering (18 May) and raised again in (water content about 60%). The differences observed between
fresh and dry samples (p b 0.05) for each collection can be
Table 2 explained by the evaporation of coumarin (from 19% to 35%
Endogenous levels of free and total coumarin (mg/kg dry weight) in Bastard depending on the different collection periods) liberated from
balm leaves (subsp. melissophyllum) during the phenological cycle.a its precursor during the drying process. At this regard, drying
Date of Fresh leaves Dry leaves
is reported to inducesù the formation of coumarin on the sur-
collection face of the plant leaves, as a response to traumatic injury during
Free Total Free Total the wilting process [22]. Therefore, the total coumarin content
3 May 7076 14,392 2537 11,125 resulted significantly (p ≤ 0.05) higher in fresh samples.
18 May 3815 6635 1168 5363 Comparing the levels of coumarin in bastard balm leaves
30 May 5380 8861 1330 6102
obtained with and without acidic treatment, we can notice
14 June 6181 9742 2216 6297
28 June 5536 7984 1816 5645 the following points: 1) as can be seen in the chromatogram
12 July 2617 4548 691 3091 reported in Fig. 1b, the bound precursor is hydrolysed effective-
2 August 2843 4588 363 3143 ly by acidic treatment; 2) there is a significant increase in total
Mean 4779 8107 1446 5824 coumarin content both in fresh (p b 0.05) and dry samples
a
Data are means of four determinations. (p b 0.001) after treatment with hydrochloric acid. This can be
1220 F. Maggi et al. / Fitoterapia 82 (2011) 1215–1221
due to the high amounts of the bound precursor (mean of 40
and 80% in fresh and dry samples, respectively) found in bas-
tard balm leaves where it is stored in the vacuole of cells. Con-
sequently, the content of coumarin after acidic treatment
showed a mean value from ~2- to 4-fold higher than in fresh
and dry leaves without hydrolysis, respectively (Table 2).
These results are in contrast to those reported for cassia and
Ceylon cinnamon, and green tea leaves [4,6], where it was
found that coumarin mostly occurred in its free form. On the
other hand, our results are in accordance with those observed
in woodruff (Asperula odorata), where only a minor part of
the total coumarin occurred in its free form, being 88% present
as bound precursor [4]; and 3) after the drying process, free
coumarin decreased under a factor of about 3 orders of magni-
tude (Table 2), suggesting that this process influenced the con-
tent of coumarin in the final product.
3.3. Coumarin as chemotaxonomic marker
As reported above, bastard balm occurs in Italy with two
subspecies: the subsp. melissophyllum, occurring in the North-
Western and central Italy; and the subsp. albida occurring in
southern Italy and Islands. So far, the classification of these sub-
species was exclusively morphology-based, being the density
of glandular trichomes, the dimension of the leaves and the
number of teeth on each side used as the main discriminant
characters.
Previously, by using SPME coupled to GC-FID and GC-MS,
we found that headspace levels of coumarin in the subsp. albida
were significantly lower than in the subsp. melissophyllum [23],
suggesting that this molecule can be used as a chemotaxonom-
ic marker.
Therefore, another aim of this work was to discriminate
the two subspecies by detecting significantly different con-
centrations of this marker compound in the plant matrix,
hence to support the botanical classification.
The total coumarin content detected in dry leaves of the
subsp. albida were 19 mg/kg in the population from Calabria,
26 mg/kg in the population from Basilicata and 35 mg/kg in
the population from Sicily, thus far lower (about 220-fold)
than the mean content detected in dry leaves of the subsp.
melissophyllum (5824 mg/kg) (p b 0.001). These results are in
agreement with the chemical differences previously reported
at headspace level between the two subspecies [23], supporting
the use of coumarin as a chemotaxonomic marker for bastard
balm. Thus, the subsp. albida resulted to have concentrations
of coumarin in line with those reported for Ceylon cinnamon
(Cinnamomum zeylanicum) bark essential oil (40 mg/kg) and
blue-white clover seeds (Trigonella caerulea) (37 mg/kg) [4].
Therefore, this subspecies should be preferred to the subsp.
melissophyllum in the use as flavoring in food products and
beverages.
4. Conclusions
The present work reports the first quantification of cou-
marin in bastard balm (M. melissophyllum). In this way, the
EFSA ‘Compendium of botanicals that have been reported to
contain toxic, addictive, psychotropic or other substances of
concern’ can be updated. A simple, fast and low-cost HPLC
method for determination of endogenous coumarin levels
has been applied. It resulted suitable for a rapid screening
of numerous plant samples to be analyzed.
This plant (notably the subsp. melissophyllum) was found to
contain among the highest levels of coumarin detected so far.
Hence, if bastard balm is regarded as a foodstuff, the coumarin
concentration in the leaves is much higher than the maximum
level permitted posing health risk to humans. However, the
plant was traditionally used under decoction or infusion as di-
gestive, hence it might be treated as a beverage. Assuming a
mean content of coumarin of 6 g/kg, as detected in our study,
only 750 mg of dried leaves in infusion with 500 ml water are
needed to equal the maximum limits of coumarin in beverages
(2 mg/l). On the other hand, about 1 g of dried leaves, if eaten,
are enough to reach a daily coumarin intake corresponding to
the TDI.
The changes in the content of coumarin during the pheno-
logical cycle were investigated highlighting the early stages
of the cycle and the fruiting time as the most appropriate pe-
riods for the production of the molecule in the plant. Thus,
coumarin levels can vary widely (up to more than 3-fold) be-
tween different sample harvesting times.
The precursor molecule, o-coumaric acid glucoside, was
detected in bastard balm leaves. The results indicated that cou-
marin occurred as much as in its bound and free form, and that
drying processing was found to influence the content of cou-
marin in the final product leading to a loss of the volatile in
its free form due to evaporation.
Coumarin turns out as a molecule of chemotaxonomic value,
to be successfully used in the support of the taxonomy of bas-
tard balm since its amount clearly differentiated the subsp.
melissophyllum with respect to the subsp. albida. Thus, this
work represents the first phytochemical approach using HPLC
method as an effective tool to distinguish the two chemotypes
of bastard balm, giving a rapid and unequivocal discrimination
using coumarin as marker compound.
Finally, it is recommended that traditional consumers use
prudentially bastard balm leaves with low coumarin levels,
corresponding to dry material collected at the latter stages
of the plant cycle and belonging to the subsp. albida.
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